Antibacterial potential of essential oils of Zataria multiflora and Mentha piperita, micro- and nano-formulated forms

Background and Objectives: Plant-derived essential oils (EOs) shave many usages in health and medicine, such as antibacterial agents. The aim of this study was the improvement of antibacterial activities of two EOs using nanotechnology. Materials and Methods: Antibacterial activity was investigated on four important human pathogenic bacteria using the 96-well plate microdilution method, a quantitative approach. Eleven formulations were prepared using each of the EOs. Eventually, the best nanoformulation with the smallest particle size and polydispersive indices (PDI and SPAN) was selected using each EO for further investigations. Moreover, two microemulsions with similar ingredients and the same portion in comparison with two selected nanoemulsions were also prepared. Antibacterial activity of each EO was compared with its micro- and nano-emulsions. Results: The antibacterial efficacy of Zataria multiflora EO (ZMEO) was significantly better than Mentha piperita EO (MPEO). Besides, the antibacterial activity of nanoemulsion of ZMEO with a particle size of 129 ± 12 nm was significantly better than no- and micro-formulated forms of ZMEO. Interestingly, the efficiency of MPEO nanoemulsion (160 ± 25 nm) was also significantly better than MPEO and its micro-formulated form. Conclusion: Regardless of the intrinsic antibacterial property of two examined EOs, by formulating to nanoemulsion, their efficiencies were improved. Nanoemulsion of ZMEO introduced as an inexpensive, potent and green antibacterial agent.


INTRODUCTION
Nanotechnology defined as targeted manipulations of materials in nanoscale for obtaining size-depen-dent features or functions (1). The most common nanomaterials are metallic nanoparticles (2), polymeric nanoparticles (3), lipidic nanocarriers (4) and nanoemulsions (5). Nanoemulsions are submicron-sized emulsions (generally 1-200 nm) in which two immiscible liquid (aquatic and oil phases) are mixed to form a single-phase, using one or more surfactants with or without using external energy such as ultrasound or probe homogenizer (6). The repeatable and straightforward manners for preparation of nanoemulsions with small and monodisperse particles has led to the widespread use of those in agricul-ture (7), health (8) and medicine (9).
Essential oils (EO)s are natural aromatic compounds derived from different parts of plants such as bark and stem (10). EO shave been widely used in health and medicine for many years, e.g., for larvicidal activity (11), antifungal purpose (12), antiparasitic research (13) and antibacterial effect (14).
In this research, antibacterial activities of two medicinally important plant-derived EOs, including Zataria multiflora (ZMEO) and Mentha piperita (MPEO), were investigated using the microdilution method. Targeted bacteria were some of the important pathogens, i.e., Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae. Moreover, we tried to improve the antibacterial activities of the EOs by formulating them into the nanoemulsions dosage form. Also, by preparing the microemulsion of ZMEO and MPEO, examined the effect of particle size on the antibacterial effect.
The procedure of GC-MS analysis. The EOs components were identified using GC-MS analysis, which described in our previous report (5).

Investigation of antibacterial activity of EOs.
96-well plate microdilution method was used for determining the growth inhibitory effect of EOs against target bacteria with slight modification (15). Briefly, new bacterial colonies were dissolved in a defined amount of nutrient broth (2×; Concentration twice as standard) to reach 0.5 McFarland (1.5 × 10 8 CFU/mL) turbidity in 630 nm by 0.08 to 0.1 optical density. Then 20 and 80 µL of the bacterial suspension and the nutrient broth, respectively, were added to each well of the plate using an 8-channel pipette.
A stock solution of each EO was prepared by dissolving in normal-saline (NS) at a concentration of 4000 µg.mL -1 (noted, at a higher level, EOs did not dissolve in NS). Then, serial dilutions of ZMEO and MPEO were prepared with a two-dimensional dilution of stock solutionin NS for developing concentration ranges of 4000-62.5 µg.mL -1 . By the addition of 100 µL from serial dilutions to each well, the concentration of EOs finally fixed at 2000, 1000, 500, 250, 125, 62.5 and 31.25 µg.mL -1 . Plates were then incubated at 37°C for 24 hours, and then absorption of wells was read at 630 nm using a plate reader (Synergy HTX-Multi-Mode Reader, USA). The tests were repeated three times, and in each replicate, six wells considered as control and blank groups. For the control groups, 20, 80 and 100 µL form the bacteria suspension, the nutrient broth, and NS respectively, was added to each well. Blank wells contained nutrient broth and NS (100: 100 µL). Using the following equation, growth (%) of bacteria at each concentration was determined.
Growth (%) = A sample -A blank / A Control -A blank × 100 *A: Mean absorption Preparation of EOs nanoemulsions. Many components of EOs are volatile. Thus, the spontaneous method was used for preparing nanoemulsions (16). A defined amount of ZMEO or MPEO (separately) and tween 20 were entirely mixed at room temperature to form a homogenous solution (500 rpm, 10 min). NS was then added dropwise to the mixture up to the desired volume (i.e., 5000 µL). The prepared mixture was stirred at 1500 rpm for 30 min. For the preparation of emulsions, eleven amounts of tween 20 (as a surfactant) and NS (as aqueous phase) wereused (0-50 and 4940-4995 µL, respectively). The highest concentration of each EO, which its growth inhibitory activity on all targeted bacteria was close together, was chosen for the preparation of emulsion. These amounts for ZMEO and MPEO included 250 and 500 µg.mL -1 ( Fig. 1A and B).
Because the emulsions should be diluted during the antibacterial test, the formulations were made using 4× more EOs. Used amounts of ZMEO and MPEO in their formulations (with the volume of 5000 µL) were fixed at 5 and 10 µL respectively. In other words, by addition of 50 µL of such emulsion into each well containing 150 µL other substrates (i.e., bacteria, NS and nutrient broth), the concentration of ZMEO and MPEO fixed at the mentioned concentrations (250 and 500 µg.mL -1 , respectively). http://ijm.tums.ac.ir Characterization of prepared emulsions: analyses of size. The mean diameter of particle sizes (PS), polydispersity index (PDI) and particle size distributions (SPAN) of prepared emulsions were determined using nanoparticle size analyzer apparatus (SZ-100 series, HORIBA Scientific, Japan). For each of EO, nanoformulation with lowest PS, PDI and SPAN was selected as optimum nanoemulsion for antibacterial tests.
Investigation of size effect on antibacterial activity. Another study was designed to investigate the impact of emulsion size on antibacterial activity. For this purpose, two microemulsions with similar components (with the same portion) to optimized nanoemulsions with bigger particle sizes (PS, PDI and SPAN) were also prepared. In the preparation procedure of those emulsions, were named microemulsion, NS was added at one-shot instead of dropwise addition.

Comparison of antibacterial activity of EOs with their nano/micro-emulsions.
Comparisons of antibacterial activities of ZMEO and MPEO with their nano/micro-emulsion were investigated at 250 and 500 µg.mL -1 , respectively, as detailed in previous sections with slight modifications. In the first step, 50 µL from each EO and it's micro-and nano-emulsion was added to wells, separately. Then 80, 50 and 20 µL of nutrient broth (2×), NS and the prepared bacteria suspension were added to each well, respectively. Then plates were incubated at 37°C for 24 hours and absorption was read at 630 nm using a plate reader. Using equation 1, the bacterial growth inhibitory activity of each sample was calculated.

Bacteria growth inhibitory activity of EOs.
Bacterial growth inhibitory effects of ZMEO on targeted strains are demonstrated in Fig. 1(A). Bacteria were exposed 24 hours with a concentration range of each EO, separately (i.e., 31.25-2000 µg.mL -1 ). IC50 of ZMEO against S. aureus, E. coli, P. aeruginosa and K. pneumoniae were observed at 129.41, 155.2, 717.58 and 140.94 µg.mL -1 , respectively. Lower and upper confidence limits of mentioned IC50 are also given in Fig. 1. Interestingly, at concentrations of 1000 and 2000 µg.mL -1 growth of S. aureus, E. coli and K. pneumoniae reduced to ~ 0%. Furthermore, the antibacterial properties of MPEO are also shown in Fig. 1(B). The inhibitory effect of MPEO (IC50) against S. aureus, E. coli, P. aeruginosa and K. pneumoniae was achieved as 27482, 18971, 6870 and 5000 < µg.mL -1 , respectively. For other factors, including lower and upper confidence limits, see Fig. 1.

Prepared emulsions and selecting optimum nanoemulsions. Details of 22 prepared ZMEO and
MPEO emulsions are given in Table 1. All emulsions were prepared at defined volume (5000 µL) using different amounts of tween 20 and NS (as an aqueous phase). Noted that, due to the lower antibacterial activity of MPEO in comparison with ZMEO, the used amount of that was fixed at 10 µL (instead of 5 µL).
To select the optimum nanoformulation, PS should be less than 200 nm, and distribution factors must be within acceptable limits, i.e., PDI < 0.7 and SPAN < 1. In this regard, one formulation form each EO was selected as optimum nanoemulsions. They called ZM3 NF, and MP5 NF and their DLS analyses are illustrated in Fig. 2 (A and C), respectively.
Comparison of size of the prepared micro-and nano-emulsions. For evaluating size effect on antibacterial activity, microemulsions (with the same ingredients and similar amounts) with bigger PS, PDI and/or SPAN in comparison to the selected nanoemulsions were also prepared. They called ZM3 MF and MP5 MF (see Fig. 2 (B and D)). PS of ZM3 MF and MP5 MF were 580 ± 42 and 230 ± 28 nm, respectively. Other size parameters of ZM3 MF include PDI and SPAN, were 4.530 ± 0.9 and 5.58 ± 1.0, respectively. Furthermore, the PDI value for MP5 MF was 0.349 ± 0.2, and SPAN was 2.043 ± 0.81.    ity of each EO in comparison with its micro-and nano-emulsions. Fig. 3. compares the antibacterial activity of ZMEO with its micro-and nano-emulsions at a concentration of 250 µg.mL -1 . As details show, the growth of all bacteria exposed to ZM3 NF was significantly lower than ZM3 MF and ZMEO (one-way ANOVA, p < 0.05). However, no significant difference was seen between ZMEO and ZM3 MF (independent sample t-test, p > 0.05). Additionally, no significant difference (independent sample t-test, p > 0.05) was viewed between the ingredient of micro/nano-emulsion (ZM3 F(-EO)) and control group, which implied, had no significant impact on the growth of bacteria.

fig. 1. Bacteria growth inhibitory activity of ZMEO (A) and MPEO (B) and related factors
From Fig 4, the inhibitory effect of MP5 NF was significantly higher than its correspond microemulsion (MP5 MF) and non-formulated EO (MPEO) (one-way ANOVA, p < 0.05). Also, ingredients used for the preparation of nano/micro-emulsion had no significant impact on the growth of bacteria in comparison with the control group (independent sample t-test, p > 0.05).

DISCUSSION
Comparing obtained IC50s of ZMEO and MPEO on target bacteria, the antibacterial activity of ZMEO was significantly better than MPEO on all examined bacteria (Independent sample t-test, p < 0.05). Due to the high efficiency of ZMEO to control at least threeimportant types of bacteria, it introduced as a potent antibacterial substrate.
From the literature, some papers have been found on investigating the antibacterial activity of major components of MPEO and ZMEO. For instance, MIC of menthol (major parts of MPEO) against S. aureus were reported as 0.62 and 0.63 mg.mL -1 and against E. coli was 2.50 and 1.25 mg.mL -1 (21). Moreover, the antibacterial activities of thymol and carvacrol have been reported frequently, e.g., their MIC on Streptococcus salivarius was observed at 5 and 2.5 mg.mL -1 , respectively (22). Furthermore, the MIC of thymol on S. aureus and E. coli were reported as 0.31 and 5.00 mg.mL -1 , respectively (21). The fact that the main components of MPEO and ZMEO have antibacterial properties is a reasonable justification for confirming the antibacterial properties of the EOs. However, due to differences in the method of evaluation of antibacterial activity in the mentioned studies and this study, the effect of the main components on the antibacterial activity of the two essential oils needs further investigation.
Nowadays, it is accepted that the encapsulation of EO at the nanoscale (1-200 nm) leads to enhancing the physical stability of bioactive compounds and increasing their bioactivity (24,25). However, another possible mechanism for improvingthe performance of nanoemulsion compared with EO or microformulations is related to better dispersion of EO droplets in the water phase (26). So, higher contact between bacterial cells and EO droplets is expected at alower size (15). Furthermore, in this study, nanoemulsion and microemulsion were made using similar components with the same portion. Thus, the only difference between them was size. It seems small PS and better monodispersity of the nanoemulsions helped them to penetrate the bacterial cells better and damage the bacterial cell wall (in comparison with micro-and non-formulated EO) (27). Similar results with the present study can be found, i.e., better performance of nanoemulsion in comparison with non-formulated EO. For instance, MIC (%) of nanoemulsion of Lemon myrtle significantly lower than EO on S. aureus (0.062 and 0.156), Listeria monocytogenes (0.031 and 0.156), and E. coli (0.25 and 0.625) (24). Antibacterial activity (MIC%) of nano emulsion (NE) of clove EO on targeted bacteria, including Bacillus subtilis, Proteus vulgaris, S. aureus, P. aeruginosa and K. pneumoniae were reported as 0.080, 0.085, 0.075, 0.300 and 0.250, respectively, while MIC of EO was significantly higher, i.e., 0.130, 0.130, 0.130, 0.500 and 0.400, respectively (28). Antimicrobial activity of NE and non-formulated EO of Cymbopogon flexuosus against different microorganism were reported as Candida albicans (0.28 and 1.22 mg/mL), Cryptococcus grubii (0.28 and 0.58 mg/mL), P. aeruginosa (11.33 mg/mL and Not active), and S. aureus (0.58 and 0.58 mg/mL) (29).

CONClUSION
In this research it was confirmed that by formulating two examined EO into nanoemulsion, their antibacterial activity has improved. Furthermore, the antibacterial activity of nanoemulsion is significantly better than its microemulsion (with the same ingredients and amounts). Moreover, the nanoemulsion of Zataria multiflora EO is introduced as a new antibacterial substrate due to its proper potency and green components.